Dihydrofolate reductase is the site of action of several important drugs including trimethoprin, pyrimethamine and the most widely used of the cancer chemotherapeutic agents, methotrexate. Thus, interest continues unabated in attempts to understand the mechanisms by which these inhibitors bind to the target enzymic receptor with such high affinity. In fact, there is mounting evidence that the binding of normal cosubstrates as well as drugs to dihydrofolate reductase is a complex process and a variety of protein conformations with different affinities and kinetic properties have been detected. Our studies on the activation of the animal reductases suggest similar conformational changes in the protein accompanied by changes in binding affinities are involved. Therefore the relationship of a specific cysteine residue in the NH2-terminal sequences of these enzymes to the activation and mechanism of action of animal dihydrofolate reductases is the basis of this continuing study since it appears to be related to both the conformational state of the enzyme and the binding affinities and not to the enzymic mechanism per se.